Differential Protein Pathways in 1,25-Dihydroxyvitamin D3 and Dexamethasone Modulated Tolerogenic Human Dendritic Cells

Tolerogenic dendritic cells (DC) that are maturation-resistant and locked in a semimature state are promising tools in clinical applications for tolerance induction. Different immunomodulatory agents have been shown to induce a tolerogenic DC phenotype, such as the biologically active form of vitamin D (1,25(OH)₂D₃), glucocorticoids, and a synergistic combination of both. In this study, we aimed to characterize the protein profile, function and phenotype of DCs obtained in vitro in the presence of 1,25(OH)₂D₃, dexamethasone (DEX), and a combination of both compounds (combi). Human CD14⁺ monocytes were differentiated toward mature DCs, in the presence or absence of 1,25(OH)₂D₃ and/or DEX. Cells were prefractionated into cytoplasmic and microsomal fractions and protein samples were separated in two different pH ranges (pH 3–7NL and 6–9), analyzed by 2D-DIGE and differentially expressed spots (p < 0.05) were identified after MALDI-TOF/TOF analysis. In parallel, morphological and phenotypical analyses were performed, revealing that 1,25(OH)₂D₃- and combi-mDCs are closer related to each other than DEX-mDCs. This was translated in their protein profile, indicating that 1,25(OH)₂D₃ is more potent than DEX in inducing a tolerogenic profile on human DCs. Moreover, we demonstrate that combining 1,25(OH)₂D₃ with DEX induces a unique protein expression pattern with major imprinting of the 1,25(OH)₂D₃ effect. Finally, protein interaction networks and pathway analysis suggest that 1,25(OH)₂D₃, rather than DEX treatment, has a severe impact on metabolic pathways involving lipids, glucose, and oxidative phosphorylation, which may affect the production of or the response to ROS generation. These findings provide new insights on the molecular basis of DC tolerogenicity induced by 1,25(OH)₂D₃ and/or DEX, which may lead to the discovery of new pathways involved in DC immunomodulation.